410. Gene expression analysis in endometriotic lesions using laser capture microdissection

2008 ◽  
Vol 20 (9) ◽  
pp. 90
Author(s):  
L. Fu ◽  
J. E. Girling ◽  
P. A. W. Rogers
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Laser Capture Microdissection ◽  
Stromal Cells ◽  
Expression Profiles ◽  
Specific Gene ◽  
Rt Pcr ◽  
Normal Endometrium ◽  
Rna Quality ◽  
Laser Capture

Previous studies examining gene expression profiles in normal endometrium and endometriotic lesions have used RNA extracted from whole tissue samples. Results from these studies can be difficult to interpret as they reflect expression averaged across several different cell types that may be functionally quite different. The aim of this study was to establish laser capture microdissection (LCM) as a technique to examine gene expression in stromal and epithelial cells from normal and ectopic endometrium. We hypothesised that genes associated with inflammation would be elevated in cells from endometriotic lesions. Full thickness uterine samples were collected during abdominal hysterectomy from normal cycling premenopausal women. Endometriotic lesions were collected during abdominal laparoscopy. Samples were either frozen in OCT or stored in RNAlater for 12 h before freezing. Tissues were immunostained with an antibody against CD10 to identify ectopic endometrial stromal cells before LCM. Endometrial epithelial and stromal cells were collected using the PALM MicroLaser System. RNA quality was accessed using Experion. TGFβ1, MMP1, αSMA, SMAD2 and NFκB mRNA was analysed using real-time RT–PCR. Of the endometriotic samples stored in OCT (n = 58), only 14% (n = 8) had visible endometrial glands. Of these, only 37% (n = 3) had RNA of an acceptable quality for further analysis. However, RNA quality and quantity were dramatically improved in 3 of 5 samples collected in RNAlater. In preliminary studies, expression of TGFβ1 and αSMA mRNA was elevated in endometriotic lesions in comparison to the normal endometrium, whereas NFκB expression did not change. We have shown that RNAlater solution is useful to preserve RNA quality for small clinical endometriotic samples and that immuno-guided LCM-generated homogenous cell populations coupled with real-time RT–PCR can provide valuable insights into cell and disease-specific gene expression in endometriotic lesions.

scholarly journals Cell-type diversity and regionalized gene expression in the planarian intestine revealed by laser-capture microdissection transcriptome profiling

2019 ◽  
Author(s):  
David J. Forsthoefel ◽  
Nicholas I. Cejda ◽  
Umair W. Khan ◽  
Phillip A. Newmark
Keyword(s):  
Gene Expression ◽  
Laser Capture Microdissection ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Cell Types ◽  
Goblet Cells ◽  
Intestinal Cell ◽  
Specific Gene ◽  
Cell Type ◽  
Laser Capture

AbstractOrgan regeneration requires precise coordination of new cell differentiation and remodeling of uninjured tissue to faithfully re-establish organ morphology and function. An atlas of gene expression and cell types in the uninjured state is therefore an essential pre-requisite for understanding how damage is repaired. Here, we use laser-capture microdissection (LCM) and RNA-Seq to define the transcriptome of the intestine of Schmidtea mediterranea, a planarian flatworm with exceptional regenerative capacity. Bioinformatic analysis of 1,844 intestine-enriched transcripts suggests extensive conservation of digestive physiology with other animals, including humans. Comparison of the intestinal transcriptome to purified absorptive intestinal cell (phagocyte) and published single-cell expression profiles confirms the identities of known intestinal cell types, and also identifies hundreds of additional transcripts with previously undetected intestinal enrichment. Furthermore, by assessing the expression patterns of 143 transcripts in situ, we discover unappreciated mediolateral regionalization of gene expression and cell-type diversity, especially among goblet cells. Demonstrating the utility of the intestinal transcriptome, we identify 22 intestine-enriched transcription factors, and find that several have distinct functional roles in the regeneration and maintenance of goblet cells. Furthermore, depletion of goblet cells inhibits planarian feeding and reduces viability. Altogether, our results show that LCM is a viable approach for assessing tissue-specific gene expression in planarians, and provide a new resource for further investigation of digestive tract regeneration, the physiological roles of intestinal cell types, and axial polarity.

213 ANALYSIS OF STEROID HORMONE RECEPTOR GENE EXPRESSION IN THE BOVINE OVARY AFTER LASER-CAPTURE MICRODISSECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 264
Author(s):  
R. Kenngott ◽  
G. Palma ◽  
M.J. Wendl ◽  
M. Vermehren ◽  
F. Sinowatz
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Real Time ◽  
Laser Capture Microdissection ◽  
Real Time Pcr ◽  
Quality Indicator ◽  
Estrogen Receptor Alpha ◽  
Qrt Pcr ◽  
Theca Interna ◽  
Laser Capture

Developmental processes in complex organs like the ovary are difficult to study in terms of a biochemical and molecular biological analysis. Laser-assisted microdissection allows the efficient and precise capture of single cells or groups of cells of an organ within the context of time and space and permits their subsequent molecular characterization. Together with real-time PCR techniques, it is now feasible to study gene expression in defined cell populations of complex tissues, but it is essential to create standards optimized for fixation, preparation, and isolation of RNA, reverse transcription reaction, and real-time PCR protocol for every tissue of interest. The aim of our study was to develop protocols for a precise analysis of estrogen receptor alpha (ER-α) and progesterone receptor (PR) in defined compartments of the ovary (granulosa cells, theca interna cells, zona vasculosa, and zona parenchymatosa of the stroma). Additionally, the receptor proteins were localized by immunohistochemistry. A special focus was put on the question of how formalin fixation and paraffin embedding influences the quality of the isolated RNA from microdissected material, which was used for quantitative reverse transcription-PCR (qRT-PCR). Quality and quantity of total RNA extracted from formalin-fixed, paraffin-embedded (FFPE) sections and from material immersed in RNAlater® (Ambion, Foster City, CA, USA) was checked using an Experion automated electrophoresis system (Bio-Rad, Munich, Germany). The RNA quality indicator for microdissected material was between 6 and 7, and for RNAlater® material was 9 or better. Online qRT-PCR using the iCycler SYBR GreenTM protocol (Bio-Rad) was performed in a 96-well plate. Primer pairs were chosen to generate PCR products between 100 bp (ER-α) and 140 bp (PR), as RNA recovered from FFPE-laser microdissected material was expected to be considerably fragmented. Using GenEx software (BioEPS, Freisling, Germany), we showed that the expression of mRNA for PR was much stronger in the theca interna than in the 3 other compartments. Estrogen receptor alpha, on the other hand, was nearly exclusively expressed in the zona parenchymatosa and zona vasculosa of the stroma. Our results show that cells obtained after laser microdissection from FFPE ovarian material can be successfully used for subsequent real-time PCR, despite the fact the RNA quality indicator number of the isolated RNA was usually comparatively low. The data of our immunohistochemical analysis support the expression data of our RNA studies. In conclusion, laser-capture microdissection in combination with quantitative PCR is a reproducible and reliable technique for quantification of a small number of cells from FFPE material. We gratefully acknowledge the continuous support by the DFG-Graduiertenkolleg 1029 and the BMBF (ARG 08/013).

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